WO2014011005A1 - Cyanine fluorescent probe, method for detecting zinc ion using same and method for preparing same - Google Patents

Abstract

The present application relates to a cyanine fluorescent probe, to a method for detecting zinc ion using the cyanine fluorescent probe, to a method for preparing the cyanine fluorescent probe, to a fluorescent sensor comprising the cyanine fluorescent probe and to a cyanine compound.

Description

Cyanine-based fluorescent probe, zinc ion detection method, and preparation method thereof

The present application relates to a cyanine-based fluorescent probe, a zinc ion detection method using the cyanine-based fluorescent probe, a method for producing the cyanine-based fluorescent probe, a fluorescent sensor including the cyanine-based fluorescent probe, and a cyanine-based compound.

Zinc ions are essential components in various biological processes and are often present in tightly bound forms in proteins. In addition, mobile intracellular zinc ions are also present in various human tissues, including the brain, intestine, pancreas, and retina. Moreover, zinc ions are known to play an important role in the regulation of apoptosis and are released from intracellular metal proteins during this process. Diseases resulting from free zinc metabolism are closely associated with many physiological conditions such as Alzheimer's disease, epilepsy, Parkinson's disease, stroke, and infant diarrhea. Although the molecular basis of the involvement of mobile zinc ions in pathophysiological processes has been extensively studied, the fundamental questions remain, in particular, questions regarding the role played by mobile zinc ions in the regulation of apoptosis. have. Therefore, the development of improved methods for quantifying and visualizing the mobile zinc ions is very important.

Fluorescent sensors have been a powerful tool for observing in vitro and in vivo levels of biologically related species and for understanding their function. Zn ²⁺ selective fluorescent sensors have been used to obtain a great deal of information about zinc biology. In most of the above fields, the strategy used to develop sensors for zinc ions is zinc in fluorophores such as fluorescein, coumarin, and 4-nitrobenzooxadiazole. There has been a focus on conjugating specific receptors. Some fluorescent zinc sensors have been used to image endogenous Zn ²⁺ in living cells and hippocampus slices, and more interestingly, visible light active fluorescent probes have been used to track endogenous Zn ²⁺ during zebrafish development. .

Despite the attractive properties of these probes, currently developed fluorescent zinc ion sensors have some serious drawbacks due to the inherent signal contamination caused by autofluorescence, high light scattering, and relatively low binding affinity. Have In particular, strong background signals, in which relatively small wavelength shifts occur at maximum absorption and emission of the probe due to the combination with Zn ²⁺ , are a serious problem. In order to overcome the limitations of these serious problems, high zinc-selective fluorescent probes that emit at long wavelengths are more desirable for detecting levels of endogenous zinc ions in living cells and organisms, which may cause the probe to have a low autofluorescence background. And less damage to biological samples. In addition, the ratiometric method, where the analytical concentration is determined by measuring the ratio of absorbance or fluorescence intensity at two wavelengths, has a built-in correction that eliminates ambient effects and increases signal fidelity. ). To date, several ratiometric fluorescent probes that emit at long wavelengths have been designed for in vivo detection of endogenous Zn ²⁺ .

In recognition of these limitations associated with previously developed methods, the inventors aim to develop a highly selective long wavelength-absorbing fluorescent Zn ²⁺ sensor that exhibits large emission wavelength variations in response to zinc ions in living cells and organisms. The research has been carried out. Despite the need for high sensitivity and high selectivity detection probes for detection of zinc in living cells and organisms, much research has not yet been conducted on high sensitivity and high selectivity zinc fluorescent probes. For example, "a method for simultaneously imaging sodium / calcium activity using two-photon fluorescent probes of different fluorescent colors and a method for preparing a two-photon fluorescent probe [Korea Patent Publication No. 2012-00013627]", etc. There has been a study on how to image sodium / calcium activity. However, very little research has been reported on high-sensitivity and high-selectivity zinc fluorescent probes.

The present application is a zinc-selective probe that emits light at a long wavelength, and has a high sensitivity and high selectivity to zinc, and reacts with endogenous zinc in living cells and organisms to show a change from blue to red at the maximum emission wavelength and a large short wavelength shift. As a cyanine derivative for detecting zinc ions, a cyanine fluorescent probe, a zinc ion detection method using the cyanine fluorescent probe, and a method of manufacturing the cyanine fluorescent probe are provided.

However, the problem to be solved by the present application is not limited to the above-mentioned problem, another problem that is not mentioned will be clearly understood by those skilled in the art from the following description.

The first aspect of the present application can provide a cyanine-based fluorescent probe comprising a compound represented by the following formula (1):

[Formula 1]

.

The second aspect of the present application can provide a method for detecting zinc ions in vivo using the cyanine-based fluorescent probe.

According to a third aspect of the present disclosure, a method of preparing a cyanine-based fluorescent probe, which includes reacting TMPA represented by the following Chemical Formula 2 with tricarbocyanine chloride, may be:

[Formula 2]

.

The fourth aspect of the present application can provide a fluorescent sensor including the cyanine-based fluorescent probe.

The fifth aspect of the present application can provide a cyanine compound represented by the following Chemical Formula 1:

[Formula 1]

.

Highly sensitive and highly selective cyanine-based fluorescent sensors can be prepared according to the present invention, which can be used to detect endogenous Zn ²⁺ ions in living cells and organisms. In the cyanine-based fluorescent probe included in the fluorescent sensor according to the present invention, when Zn ²⁺ is added to a solution of CTMPA, a color change from blue to bright red, which can be easily determined by the naked eye, occurs, and at the same time, the maximum emission of CTMPA Significantly large hyperchromic shifts from about 730 nm to about 590 nm, with a wavelength change of about 140 nm, and cell or neural globules can be detected. The CTMPA according to the present disclosure is capable of absorbing zinc ions in living cells and organisms because of useful features including large spectral shift induced by Zn ²⁺ , low fluorescence background, and high sensitivity to Zn ²⁺ . Can be used to detect. The CTMPA can be used to observe endogenous zinc ions released during apoptosis, and the probe can be used to track intact Zn ²⁺ during zebrafish development. Due to the low background and high sensitivity of the CTMPA, the CTMPA according to the present application is the first fluorescence detection probe of neuromasts in zebrafish, and can be very useful in zinc biology research. Finally, a strategy that relies on the change in the π-electron conjugation length of cyanine molecules promoted by metal ion coordination may show great potential for the creation of new cyanine probes.

1 is a schematic diagram illustrating the synthesis of fluorescent Zn ²⁺ probe CTMPA according to one embodiment of the present disclosure.

2A-2D are Zn ²⁺ (0 μM, 0.5 μM, 1.0 μM, 1.5 μM, 2.0 in HEPES buffer (10 mM, pH = 7.4) containing 10% CH ₃ CN according to one embodiment herein Absorbance and emission spectra of the appropriate CTMPA (5.0 μM) are shown using μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, and 5.0 μM).

FIG. 3 is a schematic diagram showing a given binding mode of CTMPA for Zn ²⁺ according to one embodiment of the present disclosure.

4A shows the pH profile of CTMPA in a mixture of CH ₃ CN-H ₂ O (1: 9) according to one embodiment of the present application, and FIG. 4B shows CH ₃ CN-H ₂ O according to one embodiment of the present application. Shows the change in absorbance of CTMPA in a mixture of (1: 9), and FIG. 4C shows the change in fluorescence intensity at 730 nm of CTMPA in a mixture of CH ₃ CN-H ₂ O (1: 9) according to an embodiment of the present application. will showing a λ _ex = 670 nm), Figure 4d is a _{CH 3 CN-H 2 O (} 1 in accordance with one embodiment of the present application: the CTMPA in a mixture of 9) I _590nm / I _730nm, the fluorescence intensity ratio (λ _ex = 560 nm).

5 is a schematic diagram of a given pH-response mode of CTMPA according to one embodiment of the present disclosure.

FIG. 6A illustrates hydrogen numbering according to the presence or absence of Zn ^{2+ of} CTMPA according to an embodiment of the present application, and FIG. 6B illustrates ¹ of CTMPA in CD ₃ CN during titration using Zn ²⁺ according to an embodiment of the present application. H NMR spectrum is shown. Wherein, (i) is the spectrum of the glass CTMPA, (ii) the [Zn ^2+] / 0.5 of [CTMPA]: a mixture of a spectrum of the 1, (iii) the [Zn ^2+] / a [CTMPA] 1 : The spectrum of the mixture of 1 is shown.

Figure 7 shows a ¹ H NMR spectrum of CTMPA during Zn ²⁺ titration in CD ₃ CN according to an embodiment of the present application.

8A shows an absorption spectrum of CTMPA (1 μM) using Zn ²⁺ titration in CH ₃ CN-HEPES (1: 9) (λ _ex = 560 nm) according to one embodiment of the present application, FIG. 8B The emission spectrum of CTMPA (1 μM) using Zn ²⁺ titration in CH ₃ CN-HEPES (1: 9) (λ _ex = 560 nm) according to an example of is shown.

Figure 9a is a graph showing the absorption ratio ( A _{510 nm} / A _{670 nm} ) of CTMPA (1 μM) to log ₁₀ C _{[Zn2 +] free} in the buffer according to an embodiment of the present application, Figure 9b is one embodiment of the present application of a buffer solution according to example CTMPA (1 μM) versus log ₁₀ C _{[Zn2 +]} is a graph showing the fluorescence ratio _{(I 590 nm / I 730 nm} ) of the _free.

10 is a bar graph showing metal ion selectivity of CTMPA according to one embodiment of the present disclosure.

FIG. 11 shows fluorescence images of exogenous zinc ions in (a) C2C12 and (b) NIH3T3 cells using CTMPA according to one embodiment of the present application.

12 shows fluorescence images of intact zinc ions in (a) C2C12 and (b) NIH3T3 cells released during apoptosis using CTMPA according to one embodiment of the present application.

FIG. 13 shows fluorescence images of intact zinc ions released into dead cells in (a) C2C12 and (b) NIH3T3 cells using CTMPA according to one embodiment of the present application.

14 shows (a) 24 hours, (b) 36 hours, (c) 48 hours, (d) 72 hours, and (e) 96 after fertilization incubated with CTMPA for 1 hour according to one embodiment herein. Fluorescence detection photographs of intact zinc ions in zebrafish that have elapsed time.

15 is a photograph showing detection of endogenous zinc ions in zebrafish using CTMPA according to an embodiment of the present application.

FIG. 16 is an image showing distribution of neurospheres along grouped living bodies in (A) side and (B) dorsal region according to one embodiment of the present disclosure. FIG. The supraorbital region includes preoptic (PO), and supraorbital (SO) neurospheres. The caudal-cranial region includes the ear (otic, O), the occipital (OC), the dorsal (D), and the middle (MI) neurospheres, and the posterior (P) Neurospheres are placed in the trunk area.

17A-17H show NMR and mass spectrometer spectra of compounds according to one embodiment of the present application, respectively.

Hereinafter, embodiments and examples of the present disclosure will be described in detail with reference to the accompanying drawings so that those skilled in the art may easily implement the present disclosure.

As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. In the drawings, parts irrelevant to the description are omitted for simplicity of explanation, and like reference numerals designate like parts throughout the specification.

Throughout this specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding the other components unless specifically stated otherwise. As used throughout this specification, the terms "about", "substantially" and the like are used at, or in the sense of, numerical values when a manufacturing and material tolerance inherent in the stated meanings is indicated, Accurate or absolute figures are used to assist in the prevention of unfair use by unscrupulous infringers. As used throughout this specification, the term "step to" or "step of" does not mean "step for."

Throughout this specification, the term "combination (s) thereof" included in the representation of a makushi form refers to one or more mixtures or combinations selected from the group consisting of the components described in the representation of makushi form, It means to include one or more selected from the group consisting of the above components.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B."

The first aspect of the present application can provide a cyanine-based fluorescent probe comprising a compound represented by the following formula (1):

[Formula 1]

.

The cyanine-based fluorescent probe according to the present invention has high selectivity and high sensitivity to endogenous zinc ions in living cells and organisms, and exhibits a change from blue to red and a large short wavelength shift.

In recognition of these limitations associated with previously developed methods, the inventors aim to develop a highly selective long wavelength-absorbing fluorescent Zn ²⁺ sensor that exhibits large emission wavelength variations in response to zinc ions in living cells and organisms. The research has been carried out. To achieve this goal, tricarbocyanine was chosen as the chromophore / fluorophore because it absorbs light with a large extinction coefficient, usually in the near infrared region (about 650 nm to about 900 nm). One potential difficulty with using this type of chromophore / fluorophore is that there is a polymethine π-electron system in which the modulation of the π-electron of the cyanine molecule is not easily modified between two nitrogen atoms. Often difficult.

In order to overcome this obstacle, a novel strategy can be adopted such that significant wavelength shifts occur in the maximum absorption and emission of the cyanine sites that depend on changes in the degree of conjugation of the π-electron system. Cyanine-based Zn²⁺Two factors can be considered in designing selective probes. As a first element, the probe is Zn²⁺It must contain a site showing strong and selective binding. For this purpose, the large Zn of the tris-pyridine moiety (TMPA)²⁺Tris-pyridine sites (TMPA) can be introduced into the probe due to binding affinity (see FIG. 1). As a second factor, more importantly, the sensor is Zn²⁺ The binding should include features that allow alteration of the effect of meso-substituents on the polymethine π-electron field of the tricarbocyanine chromophore / fluorophore. In this way, Zn²⁺The combination of will result in a large shift in maximum absorption and luminescence, resulting in a low fluorescence background.

Based on these criteria, Zn²⁺The fluorescent Zn expected to have a clear and reversible change in the π-electron field of the cyanine chromophore / fluorophore upon complexation of²⁺Probe CTMPA can be designed as a sensor for in vivo detection of endogenous zinc ions (FIG. 1). In the following, the results of a study exploring the manufacture of CTMPA and its in vivo application are described. Observations obtained with this effort indicate that CTMPA is Zn²⁺Induced large absorption and emission wavelength shift, low fluorescence background, and Living cells and endogenous Zn in living organisms²⁺It can be demonstrated that there are a number of useful features, including the specific applicability to track them.

According to one embodiment of the present application, the cyanine-based fluorescent probe may be used for detecting zinc ions (Zn ²⁺ ), but may not be limited thereto.

According to the exemplary embodiment of the present application, the compound represented by Chemical Formula 1 may be reacted with zinc ions (Zn ²⁺ ) to form a complex, but may not be limited thereto. For example, the cyanine-based fluorescent probe including the compound represented by Formula 1 may detect endogenous zinc ions (Zn ²⁺ ) in vivo, but may not be limited thereto.

According to an embodiment of the present disclosure, the compound represented by Chemical Formula 1 may be a color change from blue to red at the maximum emission wavelength in response to Zn ²⁺ , but may not be limited thereto.

According to the exemplary embodiment of the present application, the compound represented by Chemical Formula 1 may represent short wavelength shift in the emission wavelength and the absorption wavelength by reaction with Zn ²⁺ , but may not be limited thereto.

According to an embodiment of the present disclosure, the compound represented by Chemical Formula 1 may exhibit short wavelength shift from about 730 nm to about 590 nm at the maximum emission wavelength by reaction with Zn ²⁺ , but may not be limited thereto. have.

According to the exemplary embodiment of the present application, pyridine and meso-substituted nitrogen in the compound represented by Chemical Formula 1 (CTMPA) may be coordinated by reacting with Zn ²⁺ , but may not be limited thereto.

According to one embodiment of the present application, the compound represented by Formula 1 (CTMPA) and the Zn ²⁺ may be to form a complex in a ratio of about 1: about 1, but may not be limited thereto.

According to the exemplary embodiment of the present application, the cyanine-based fluorescent probe may detect endogenous zinc ions (Zn ²⁺ ) in vivo, but may not be limited thereto.

According to the exemplary embodiment of the present application, the cyanine-based fluorescent probe may detect zinc ions within the nM range, but may not be limited thereto. The cyanine-based fluorescent probe may be, for example, about 0.1 nM to about 1,000 nM, about 0.1 nM to about 500 nM, about 0.1 nM to about 100 nM, about 0.1 nM to about 80 nM, about 0.1 nM to about 50 nM , About 0.1 nM to about 30 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 1 nM, about 1 nM to about 1,000 nM, about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 80 nM, about 1 nM to about 50 nM, about 1 nM to about 30 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 5 nM To about 1,000 nM, about 5 nM to about 500 nM, about 5 nM to about 100 nM, about 5 nM to about 80 nM, about 5 nM to about 50 nM, about 5 nM to about 30 nM, about 5 nM to about 10 nM, about 10 nM to about 1,000 nM, about 10 nM to about 500 nM, about 10 nM to about 100 nM, about 10 nM to about 80 nM, about 10 nM to about 50 nM, about 10 nM to about 30 nM , About 30 nM to about 1,000 nM, about 30 nM to about 500 nM, about 30 nM to about 100 nM, about 30 nM to about 80 nM, about 30 nM to about 50 nM, about 50 nM to about 1,000 nM, about 50 nM to about 500 nM, about 50 nM to about 100 nM, about 50 nM to about 80 nM, about 80 nM Detects zinc ions from about 1,000 nM, about 80 nM to about 500 nM, about 80 nM to about 100 nM, about 100 nM to about 1,000 nM, about 100 nM to about 500 nM, or about 500 nM to about 1,000 nM It may be, but may not be limited thereto.

The second aspect of the present application can provide a method for detecting zinc ions in vivo using the cyanine-based fluorescent probe.

According to one embodiment of the present application, the detection method may be one capable of imaging the distribution of endogenous zinc ions in vivo, but may not be limited thereto.

According to a third aspect of the present disclosure, a method of preparing a cyanine-based fluorescent probe, which includes reacting TMPA represented by the following Chemical Formula 2 with tricarbocyanine chloride, may be:

[Formula 2]

.

According to the exemplary embodiment of the present application, the TMPA and the tricarbocyanide chloride may be reacted by heating at about 70 ° C. to about 100 ° C. under an argon atmosphere, but may not be limited thereto.

According to one embodiment of the present application, it may further include purifying the cyanine-based fluorescent probe, but may not be limited thereto.

The fourth aspect of the present application can provide a fluorescent sensor including the cyanine-based fluorescent probe.

The fifth aspect of the present application can provide a cyanine compound represented by the following Chemical Formula 1:

[Formula 1]

.

The second to fifth aspects of the present application, respectively, using the cyanine-based fluorescent probe according to the first aspect of the present invention, zinc ion detection method in vivo, a method for producing a cyanine-based fluorescent probe according to the first aspect of the present application, the cyanine-based A fluorescent sensor including a fluorescent probe, and a cyanine compound represented by Chemical Formula 1, and detailed descriptions of portions overlapping with the first aspect of the present disclosure are omitted, but the descriptions of the first aspect of the present disclosure The same may be applied to each of the second to fifth aspects of the present disclosure even if the description thereof is omitted.

Hereinafter, with reference to the embodiment and the drawings will be described in detail. However, the present application is not limited to these examples and drawings.

EXAMPLE

Example 1 Cyanine-Based Probe Derivatives and Preparation

Substances and Methods

Unless stated otherwise, the materials used were from Aldrich and were used without further purification. ¹ H NMR and ¹³ C NMR spectra were measured on a CDCl ₃ solution containing tetramethylsilane (TMS) as internal standard using Bruker AM-300 spectrometer. Mass spectra were acquired using a JMS-HX 110A / 110A Tandem mass spectrometer (JEOL). UV-VIS spectra were acquired using a Scinco 3000 spectrophotometer (1 cm quartz cell) at 25 ° C. Fluorescence spectra were recorded on an RF-5301 / PC (Shimada) fluorescence spectrophotometer (1 cm quartz cell) at 25 ° C. Deionized water was used to prepare all aqueous solutions.

Precursor synthesis for the synthesis of CTMPA including TMPCl

Thionyl chloride (15 mL) was added to 2,6-bis (hydroxymethyl) pyridine (2.0 g, 14.4 mmol) through an additional funnel at 0 ° C. over 30 minutes. After warming at room temperature, the reaction mixture was heated to reflux for 5 hours and then cooled to room temperature. Toluene (20 mL) was added slowly to the mixture to precipitate a white solid. The filtered hydrochloride was dissolved in 200 mL water and solid Na ₂ CO ₃ was slowly added to the solution until pH 7 was reached. The white solid was precipitated out, filtered and washed repeatedly with water. The crude product was recrystallized from petroleum ether to give the product as a colorless needle: ¹ H NMR (CDCl ₃ , 300 MHz) δ 7.78 (t, J = 7.5 Hz, 2H, pyridine-H ), 7.46 (d, J = 7.8 Hz, 2H, pyridine-H), 4.67 (s, 4H, Py-C H _2- ).

To a solution of 2,6-bis (chloromethyl) pyridine (1.25 g, 0.71 mmol) in 30 mL of CH ₃ CN was added phthalimide and K ₂ CO ₃ (1.32 g, 0.71 mmol). The reaction mixture was heat treated to reflux overnight and then cooled at room temperature. The mixture was filtered and concentrated under reduced pressure to give a white solid. The crude product was purified by flash column chromatography on silica gel eluting with ethyl acetate / hexanes 1: 1 (v / v) to give a white, needle-like crystal product (0.85 g, 51% yield). ): ¹ H NMR (300 MHz, CDCl ₃ ) δ 4.59 (s, 2 H, C H ₂ Cl), 5.02 (s, 2 H, -C H ₂ -phthal), 7.17 (d, J = 7.5 Hz, 1 H), 7.38 (d, J = 7.5 Hz, 1 H), 7.67 (t, J = 7.5 Hz, 1 H), 7.74-7.77 (m, 2H), 7.88-7.91 (m, 2H) ( See FIG. 17A).

Bis (2-phycoyl) amine (0.62 g, 3.11 mmol) and TMPCl (0.8 g, 2.82 mmol) were dissolved in 30 mL of CH ₃ CN. Potassium carbonate (0.83 g, 6.01 mmol) was added to the mixture, and the reaction mixture was heated to reflux for 12 hours. The crude product was purified by flash column chromatography on silica gel eluting with CH ₂ C1 ₂ / CH ₃ OH (15: 1, v / v) to give a yellow-white solid (1.1 g, 86% Yield) was provided: ¹ H NMR (300 MHz, CDCl ₃ ) δ 3.76 (s, 2 H, C H ₂ ), 3.82 (s, 4 H, 2C H ₂ ) 5.00 (s, 2 H, C H ₂ Phthal), 7.08-7.13 (m, 3H), 7.40 (d, J = 7.5 Hz, 1H), 7.51 (d, J = 7.5 Hz, 2H), 7.59 (t, J = 7.5 Hz, 3 H), 7.71-7.74 (m, 2H), 7.86-7.89 (m, 2H), 8.49 (d, 2H, pyridine-H) (see FIG. 17B).

Synthesis of TMPA

To a stirred solution of TMPP (0.85 g, 1.89 mmol) in 20 mL methanol, hydrazine hydrate (1.0 g, 20.0 mmol) was added and heat treated overnight to reflux. The solvent was removed under reduced pressure to give a white solid. The white solid was dissolved in CHCl ₃ and 200 mL of 1 M NaOH was added to the solution. The organic layer was separated, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product (orange red) was subjected to flash column chromatography on silica gel by eluting with CH ₂ Cl ₂ / CH ₃ OH (10: 1, v / v) to give a yellow-red solid product. (0.55 g, 91% yield) was provided: ¹ H NMR (300 MHz, CDCl ₃ ) δ 1.84 (br, 2 H, NH ₂ ) 3.88 (s, 2 H, CH ₂ ), 3.90 (s, 4 H , CH ₂ ), 3.94 (s, 2H, CH ₂ ), 7.11-7.17 (m, 3H), 7.45 (d, J = 7.5 Hz, 1H), 7.59-7.69 (m, 5H), 8.54 ( d, 2 H); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 47.5, 60.1, 60.1, 119.3, 120.8, 121.9, 122.8, 136.4, 136.9, 149.0, 158.7, 159.4; ESI-MS [C ₁₉ H ₂₁ N ₅ + H ⁺ ] calculated 320.1875, measured 320.1875 (see FIGS. 17C-17E).

Synthesis of CTMPA

A solution of IR-780 (0.25 g, 0.38 mmol) and TMPA (0.12 g, 0.40 mmol) in anhydrous DMF (15 mL) were heat treated at 80 ° C. to 90 ° C. for 10 hours under argon atmosphere. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography on silica gel and then eluted with CH ₂ C1 ₂ / MeOH (40: 1) to give a dark blue solid (120 mg, 32 %) Provided the desired product: ¹ H NMR (300 MHz, CDCl ₃ ) δ 1.05 (t, J = 7.5 Hz, 6 H, NH (CH ₂ ) ₂ C H ₃ ), 1.55 (s, 12 H, -CH ₃ ), 1.80-1.87 (m, 6H, NHCH ₂ C H ₂ CH _3, and cyclohexyl H ), 2.55 (t, J = 6.0 Hz, 4 H, cyclohexyl H ), 3.84 (t, J = 7.2 Hz, 4H, NHC H ₂ CH ₂ CH ₃ ), 3.90 (s, 4H, pyridine-C H _2- ), 3.94 (s, 2H, pyridine-C H _2- ), 5.06 (s, 2 H, pyridine-C H _2- ), 5.73 (d, J = 12.6 Hz, 2 H, alkene-H), 6.90 (d, J = 7.8 Hz, 2 H, Ph-H), 7.09 (t, J = 7.5 Hz, 2 H, pyridine-H), 7.24 (d, J = 7.8 Hz, 2 H, Ph-H), 7.29 (t, J = 7.5 Hz, 2 H, Ph-H), 7.43 (d, J = 7.8 Hz, 1 H, pyridine-H), 7.61 (d, J = 7.8 Hz, 2 H, Ph-H), 7.72 (d, J = 12.6 Hz, 2 H, alkene-H), 7.68-7.74 (m, 4H, pyridine-H), 7.84 (t, J = 7.8 Hz, 2 H, pyridine-H), 8.16 (br, -NH-), 8.52 (d, J = 3.6 Hz, 2 H, pyridine-H); ¹ H NMR (300 MHz, CD ₃ CN) δ 0.99 (t, J = 7.5 Hz, 6 H, NH (CH ₂ ) ₂ C H ₃ ), 1.45 (s, 12 H, -CH ₃ ), 1.74-1.82 (m, 6H, NHCH ₂ C H ₂ CH ₃ and cyclohexyl H ), 2.55 (t, J = 6.0 Hz, 4 H, cyclohexyl H ), 3.87-3.86 (m, 10 H, NHC H ₂ CH ₂ CH ₃ , 4 H and pyridine-C H _2- , 6 H), 4.93 (d, J = 5.4 Hz, 2 H, pyridine-C H _2- ), 5.80 (d, J = 13.2 Hz, 2 H, alkene -H), 7.04-7.13 (m, 4H, Ph-H), 7.15-7.20 (m, 2H, pyridine-H), 7.27-7.35 (m, 4H, Ph-H), 7.60-7.73 ( m, 6H, pyridine-H), 7.70 (t, J = 13.2 Hz, 2H, alkene -H), 7.83 (t, J = 7.5 Hz, 1H, pyridine-H), 8.47 (d × m, J = 4.8 Hz, 2 H, pyridine-H); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 11.9, 20.1, 21.6, 25.8, 29.3, 45.7, 47.7, 53.5, 60.6, 67.8, 94.8, 108.6, 120.4, 122.1, 122.8, 122.9, 123.0, 128.1, 136.9, 137.9 , 138.3, 139.8, 143.1, 148.9, 167.6, 168.6; FAB-MS [C ₅₅ H ₆₄ IN ₇ -I] ⁺ calc. 822.5223, found 822.5227 (see FIGS. 17F-17H).

CTMPA-Zn ²⁺  Determination of the apparent dissociation constant of the complex

The fluorescence spectrometer was used to determine the apparent dissociation constant ( K _d ) calculated by using the following equation;

Where R is the absorption ratio or fluorescence intensity ratio at two wavelengths, R _max is the maximum ratio, R ₀ is the ratio without addition of Zn ²⁺ , and [M ²⁺ ] _free is the ratio of _free Zn ²⁺ Concentration. The concentration of free Zn ²⁺ was controlled by using metal ion buffer [nitrilotriacetic acid (10 mM), log K (ZnNTA) = 10.66 (20 ° C., 0.1 M KNO ₃ )].

Example 2: Detection of zinc ions in living cells and organisms

Cell culture

C2C12 cells (mouse myoblast cell line) and NIH3T3 cells (mouse embryonic fibroblast cell line) were obtained from American Type Culture Collection (Manassas, VA) and culture medium [10% fetal bovine serum (FBS), penicillin 50 units / mL and 50 μg / mL of streptomycin were maintained in Dulbecco's Modified Eagle Medium (DMEM).

Fluorescence Detection of Living Cells Using CTMPA

C2C12 cells and NIH3T3 cells were seeded in 24-well plates at a density of 1 × 10 ⁴ cells per well in culture medium. After 24 hours, cells were treated with 2 μM CTMPA for 1 hour at 37 ° C. in culture medium containing 0.1% (v / v) DMSO. After washing twice with DPBS (Dulbecco's phosphate buffered saline (without calcium and magnesium)), to remove the remaining probes, cells contained 2 μM ZnCl ₂ / sodium pyrithione solution at 37 ° C. for 0.5 hour. It was further processed using DPBS. Without washing, cells were further treated with 50 μM TPEN for 0.5 h. The treated cells were imaged by confocal microscopy (LSM 510 META, Carl Zeiss, Germany, λ _ex = 543 nm).

Imaging of Apoptotic Cells Using CTMPA

C2C12 cells and NIH3T3 cells were attached in 24-well plates at a density of 1 × 10 ⁴ cells per well in culture medium. After 24 hours, cells were incubated at 37 ° C. for 1 hour in the presence of 250 μM H ₂ O ₂ . After 6 hours, cells were treated with 2 μM CTMPA at 37 ° C. for 1 hour. After washing twice with Dulbecco's phosphate buffered saline (DPBS), cells were imaged by confocal microscopy. For TPEN experiments, cells treated in the presence of 250 μM H ₂ O ₂ for 6 hours were incubated with 50 μM TPEN at 37 ° C. for 0.5 hours. After washing twice with DPBS to remove the remaining TPEN, cells were treated with 2 μM CTMPA at 37 ° C. for 1 hour. The treated cells were washed and imaged by confocal microscopy.

Tracking Distribution of Zinc Ions in Zebrafish

The zebrafish was kept at 28 ° C. and optimal reproductive conditions were maintained. For mating, the male and female zebrafish were kept in a tank at 28 ° C. with a 12 hour / 12 hour light cycle, and then scattering was induced by providing photostimulation in the morning. Nearly all eggs were fertilized immediately. All steps of zebrafish were performed in E3 embryo medium (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO ₄ , 1 mM CaCl ₂ , 0.15 mM KH ₂ PO ₄ , 0.05 mM Na ₂ HPO ₄ , 0.7 mM NaHCO ₃ , 5% to 10% methylene blue; pH 7.5). Zebrafish embryos 24, 36, 48, 72, and 96 hours after fertilization were subjected to 1 hour in E3 medium containing 0.1% (v / v) DMSO containing 2 μM CTMPA at 28 ° C. While incubated. For TPEN experiments, zebrafish was exposed to 100 μM TPEN in E3 medium at 28 ° C. for 1 hour to remove intact zinc ions in zebrafish. After washing with E3 medium to remove the remaining TPEN, the zebrafish was incubated in E3 medium containing 2 μM CTMPA at 28 ° C. for 1 hour. The treated zebrafish were washed and imaged by confocal microscopy.

Zn on the Spectroscopic Properties of CTMPA ²⁺ Effect

Due to the fact that the TMPA site of CTMPA contains three pyrimidine groups that contribute as zinc coordination sites, CTMPA has this enhanced Zn ²⁺ binding capacity. The primary amine moiety in TMPA is linked to the center of the polymethine chain of tricarbocyanine in the probe, and as a result, the coordination to Zn ²⁺ will affect the degree of conjugation of the π-electron system. This phenomenon results in observable shifts of maximum wavelengths in the absorption and emission spectra. Based on this reason, the Zn ²⁺ -specific fluorescent probe CTMPA was prepared according to the procedure shown in FIG. 1. In brief, TMPA was synthesized from 2,6-dichloromethylpyridine in 22% yield. CTMPA was prepared in 32% yield by reacting tricarbocyanine IR-780 with chloride in DMF and TMPA.

To investigate the response to Zn ²⁺ , a solution of CTMPA in HEPES (4 (2-hydroxyethyl) piperazine-1-ethanesulfonic acid) buffer (10 mM, pH = 7.4) containing 10% CH ₃ CN This was titrated using various concentrations of Zn ²⁺ . In this regard, FIGS. 2A-2D show Zn ²⁺ (0 μM, 0.5 μM, 1.0 μM, 1.5 μM in HEPES buffer (10 mM, pH = 7.4) containing 10% CH ₃ CN according to this example. , 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, and 5.0 μM) are used to show the absorption and emission spectra of the appropriate CTMPA (5.0 μM). As shown in FIG. 2, it was observed that a significant change in the absorption spectrum of CTMPA according to this example occurs with the addition of Zn ²⁺ . Regarding the color change of the solution from blue to red luminescence (inset in FIG. 2A), the addition of Zn ²⁺ was about 670 with the maintenance of an apparent isosbestic point at about 560 nm (FIG. 2A). sharp reduction of the absorption peak of CTMPA at nm (ε = 8.4 × 10 ⁴ M ⁻¹ · cm ⁻¹ ) and centered at about 510 nm (ε = 3.0 × 10 ⁴ M ⁻¹ · cm ⁻¹ ) Results in an increase in new bands. It was found that the absorption ratio of A _{510 nm} / A _{670 nm} increased linearly as a function of Zn ²⁺ concentration up to about 1: 1 ratio of Zn ²⁺ : CTMPA, indicating the formation of about 1: about 1 complex ( Inset of Figure 2a). This observation suggests that CTMPA will contribute as a colorimetric sensor for the measurement of Zn ²⁺ concentrations.

Interestingly, large short wavelength shifts in the emission spectrum of CTMPA also result in Zn²⁺It was observed to occur in the presence of. In particular, in response to such metal ions, the intensity of the near-infrared emission band of CTMPA at about 730 nm (excitation at about 670 nm) decreases (FIG. 2B) and the simultaneous increase is about 585 nm [about 510 nm]. In the emission peak of (Fig. 2C). The wavelength (approximately 560 nm) corresponding to the iso-absorption point in the absorption spectra is Zn²⁺Fluorescence intensity ratio by concentrationI                 _{590 nm}OfI                 _{730 nm}It was chosen as the excitation wavelength in measuring the dependence of. As shown in FIG. 2D, the near infrared band at about 730 nm in the emission spectrum decreases with a sharp increase in the band at about 590 nm. The observation is that the characteristic emission band of CTMPA is Zn²⁺It demonstrates that a large short wavelength shift (about 140 nm) proceeds with binding to. CTMPA-Zn²⁺ Zn strong in solution containing complex²⁺ChelatorN, N, N ', N'-Tetrakis- (2-pyridylmethyl) ethylenediamine (N, N, N ', N'-tetrakis- (2-pyridylmethyl) ethylenediamine, TPEN) added Zn to the probe²⁺Results in recovery of eigencolors and fluorescence of CTMPA, showing that the binding of is reversible. remindI                 _{590 nm}OfI                 _{730 nm}Ratio is Zn²⁺Zn until 1 equivalent of²⁺A linear correspondence with concentration was confirmed (inset in FIG. 2D). The combined result is CTMPA-Zn²⁺Of about 1: a complex of about 1 is produced andI                 _{590 nm}OfI                 _{730 nm} AndA                 _510nmOfA                 _{670 nm}Ratio of in vitro Zn²⁺It can be used to measure the concentration. 2 is 10% CH₃Zn in HEPES buffer (10 mM, pH = 7.4) containing CN²⁺(0 μM, 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, and 5.0 μM) and the absorption and emission spectra of the appropriate CTMPA (5.0 μM). Figure 2a shows an absorption spectrum [inset is Zn²⁺Before and after addition of Zn²⁺Ratio of absorbance as a function of the concentration and color change ofA                 _510nmOfA                 _{670 nm}); 2B shows the emission spectrum (λ) in the region of about 670 nm or more._ex = 670 nm); 2c shows the emission spectrum in the region of about 510 nm or more (λ)_ex = 510 nm); 2D shows the emission spectrum (λ) in the region of about 560 nm or more._ex = 560 nm) (inset is Zn²⁺Before and after addition of Zn²⁺Ratio of fluorescence intensity as a function of concentration and fluorescence change of (I                 _{590 nm}OfI                 _{730 nm})).

Zn of CTMPA ²⁺  Coupling behavior

Observations obtained in previous studies indicate that electron-donating or accepting meso-substituents have an effect on the absorption spectral properties of the polymethine dye, but these substituents do not significantly change the maximum luminescence. Indeed, the above-mentioned cyanine-based fluorescent sensor for metal ions only shows an increase and a decrease in luminescence intensity, but little or almost no movement at the maximum emission wavelength. Recently, a cyanine based metering Zn ²⁺ fluorescent probe was disclosed, which exhibited some Zn ^{2+ -promoted} red shift (about 44 nm) at maximum absorption and did not exhibit emission wavelength. In contrast, however, Zn ²⁺ promotes large short wavelength shifts in both the maximum absorption and emission of CTMPA (see FIGS. 2A-2D). Other factors than simple modification of the electron donor or acceptor properties of meso substituents appear to contribute to the luminescent properties of the CTMPA-Zn ²⁺ complex. Large luminescence wavelength changes caused by the combination of CTMPA and Zn ²⁺ may possibly be the result of the collapse of the broadly conjugated π-electron system of the polymethine chromophore / fluorophore (see below). As shown in the CTMPA-Zn ²⁺ structure shown in FIG. 3, the complexation of Zn ²⁺ with the secondary amine nitrogen in the TMPA site induces NH deprotonation and is less delocalized. Reduced delocalization will occur if it forms an imine that is cross-conjugated with the diamino-tetraene group. This transformation results in the significant destruction of the tricarbocyanine chromophore and the pull-push conjugated system, and the polymethine π-electron field of cyanine, which causes large short wavelength shifts at maximum absorption and emission. This results in shortening of.

In order to gain a better understanding of the binding mode of Zn ²⁺ to CTMPA and the resulting spectral change, a pH-dependent study of the spectroscopic changes was performed. 4a shows the pH profile of CTMPA in a mixture of CH ₃ CN-H ₂ O (1: 9) according to this example, and FIG. 4b shows CH ₃ CN-H ₂ O (1: 9) according to this example. 4C shows the change in absorbance of CTMPA in the mixture of Fig. 4C, and the fluorescence intensity change (λ _ex = 670 nm) at 730 nm of CTMPA in the mixture of CH ₃ CN-H ₂ O (1: 9) according to this example. will showing a, Fig. 4d is a _{CH 3 CN-H 2 O (} 1: 9) according to this embodiment shows a CTMPA of I _590nm / I _730nm fluorescence intensity ratio (λ _ex = 560 nm) in the mixture of. It was observed that a large blue shift occurs at a pH ranging from about 10.2 to about 11.5 at the maximum absorption and emission of CTMPA according to this example (see FIGS. 4A-4D). This significant short wavelength shift is due to the destruction of the pull-push π-conjugated system of the tricarbocyanin induced by deprotonation of the NH group in CTMPA (see FIG. 5). Deprotonation of NH in the meso-substituents of CTMPA according to this example shortens the π-conjugated system in the tricarbocyanine chromophore / fluorophore, which shows short wavelength shift at maximum absorption and emission. This result is because a large blue shift of the CTMPA increased by a combination of Zn ^2+, Zn ²⁺ in a coordination bond to the NH of the probe to lower the NH of p K _a, at about pH 7.4 the CTMPA-Zn ^{2 +} Suggestion caused by deprotonation of NH in the complex (see FIG. 3). Absorption and luminescence spectra of CTMPA according to the present embodiment remain almost unchanged between about pH 2.8 and about pH 9.7, and therefore, the probe may be applied to in vivo detection of Zn ²⁺ . It is expected.

To further investigate the mode of Zn ²⁺ binding to CTMPA according to the present application, a ¹ H NMR titration experiment was performed. The addition of Zn ²⁺ to the solution of CTMPA according to this example results in the formation of a new set of ¹ H NMR signals in addition to the set resulting from the free CTMPA (see FIGS. 6 and 7). 6a shows hydrogen numbering according to the presence or absence of Zn ^{2+ of} CTMPA according to the present embodiment, and FIG. 6b shows a ¹ H NMR spectrum of CTMPA in CD ₃ CN during titration using Zn ²⁺ according to the present embodiment. as shown, (i) is the spectrum of the glass CTMPA, (ii) the [Zn ^2+] / 0.5 of [CTMPA]: a mixture of a spectrum of the 1, (iii) the [Zn ^2+] / [CTMPA] The spectrum of the mixture of 1: 1 is shown. In addition, Figure 7 shows the ¹ H NMR spectrum of CTMPA during the Zn ²⁺ titration process of CD ₃ CN according to the present embodiment. Detailed confirmation of the ¹ H NMR signals is shown in Table 1. When the concentration ratio of CTMPA to Zn ²⁺ ([CTMPA]: [Zn ²⁺ ]) according to this example is about 1: 0.5, the two sets of signals have almost the same intensity. However, when the concentration ratio reaches about 1: 1, only ¹ H NMR signal of the CTMPA-Zn ²⁺ complex is observed. The addition of additional equivalents of Zn ²⁺ does not cause further changes in the ¹ H NMR spectrum. These results also confirm that about 1: about 1 complex between CTMPA and Zn ²⁺ according to this example is formed. The addition of about 1 equivalent of Zn ²⁺ to the CTMPA solution according to this example promotes a significantly greater downfield shift from about 8.47 ppm to about 9.31 ppm of the pyridine proton H ₁ (FIG. 2). . Moreover, Zn ²⁺ configuration results in apparent downfield displacement of H ₅ , H ₆ , and H ₇ protons. This change means that all three pyridine groups and the meso-substituted nitrogen participate in the Zn ²⁺ configuration, as shown in FIG. 6. Importantly, the chemical displacement of the alkene proton H ₁₁ shifts from about 5.80 ppm to about 5.85 ppm in the presence of Zn ²⁺ , while the alkene proton H ₁₂ shifts from about 7.70 ppm to about 7.56 by the coordination of these metal ions. Upfield shift in ppm. The observed alkene-H chemical shift further supports the conclusion that the polymethine π-electron field in CTMPA is shortened by the complexation of Zn ²⁺ with the probe.

TABLE 1

The apparent dissociation constant ( K _d ) of the CTMPA-Zn ²⁺ complex plots the fluorescence intensity ratio I _{590 nm} / I _{730 nm} or the absorption ratio A _{510 nm} / A _{670 nm} as a function of Zn ²⁺ concentration. Was measured to be about 1.2 nM (see FIGS. 8A, 8B, 9A, and 9B). FIG. 8A shows an absorption spectrum of CTMPA (1 μM) using Zn ²⁺ titration in CH ₃ CN-HEPES (1: 9) (λ _ex = 560 nm) according to the present embodiment, and FIG. 8B shows this example. The emission spectrum of CTMPA (1 μM) using Zn ²⁺ titration in CH ₃ CN-HEPES (1: 9) (λ _ex = 560 nm) according to FIG. 9A is shown in FIG. 9A. (1 μM) versus log ₁₀ C _{[Zn2 +] free} graph showing absorbance ratio ( A _{510 nm} / A _{670 nm} ), FIG. 9B shows CTMPA (1 μM) versus log ₁₀ C _[in buffer according to this Example. _It is a graph showing the fluorescence ratio ( I _{590 nm} / I _{730 nm} ) of _{Zn2 +] free} . Importantly, Zn ²⁺ Zn ²⁺ affinity of pro-di (2-picolyl) amine [di (2-picolyl) amine , DPA] (K d = 23 nm) for CTMPA according to the present embodiment in which the observation Much larger than Mars, which further indicates that all three pyridine nitrogens in the probe chelate with Zn ²⁺ . The nanomolar K _d value of CTMPA-Zn ²⁺ suggests that the probe is suitable for detecting Zn ²⁺ within the nM range.

Zn of CTMPA ²⁺  Binding selectivity

Previously developed Zn²⁺Unlike the receptors, CTMPA has three pyridine groups and Zn²⁺It contains one meso-nitrogen that acts as a coordination site. The presence of multiple coordination sites in the probe is Cd²⁺ And Zn of CTMPA relative to other metal ions²⁺It is expected to increase binding selectivity. To investigate this suggestion, comparative experiments were conducted with Na⁺, K⁺, Mg²⁺, Ca²⁺, Ag⁺, Al³⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Fe³⁺, Hg²⁺, Mn²⁺, Ni²⁺And Pb²⁺The solution of CTMPA (5 μM) and then Zn²⁺(5 μM) was added continuouslyI                 _{590 nm}OfI                 _{730 nm}It was performed by rate measurement. As shown in Figure 10, the CTMPA-Zn²⁺The emission intensity profile of the composite is not changed by the presence of the other tested metal ions, which is Zn²⁺High selectivity for CTMPA. 10 is a graphical representation of the metal ion selectivity of CTMPA, 10% CH₃CN (λ_ex570 nm) shows the fluorescence response of CTMPA (5 μΜ) to various metal ions in HEPES (10 mM, pH = 7.4). Gray bars show various competing metal ions (Na in a solution of CTMPA (5 μM)⁺, K⁺, Mg²⁺ And Ca²⁺1,000 μM for ; CD ²⁺10 μM for; After addition of 25 μM) for all other metal ionsI                 _{590 nm}OfI                 _{730 nm}Indicates a ratio. Black bars are Zn in a solution of CTMPA (5 μM) in the presence of the competing metal ions.²⁺After addition ofI                 _{590 nm}OfI                 _{730 nm}Indicates a ratio.

Intracellular Zn Using CTMPA ²⁺ Imaging of

Because of the excellent spectroscopic Zn ²⁺ binding properties of CTMPA, CTMPA will be suitable for detecting zinc ions in living cells and organisms. To test this prediction, the Zn ²⁺ bioimaging ability of the CTMPA was measured using live mammalian cells. Mouse C2C12 myoblasts and mouse NIH3T3 embryonic fibroblasts were incubated with 2 μM CTMPA for 1 hour (see FIG. 11). FIG. 11 shows fluorescence images of exogenous zinc ions in (a) C2C12 and (b) NIH3T3 cells using CTMPA according to this Example. After removal of the remaining probe by washing, the cells were treated with ZnCl ₂ / pyrithione solution (2 μM) and incubated for 0.5 hours. As a result of fluorescence imaging of exogenous zinc ions, cells cultured with CTMPA alone show very weak fluorescence, while cells treated with Zn ²⁺ and the probe show strong fluorescence, which is a cell membrane-permeable zinc ion chelator TPEN It is significantly weakened by pre-incubation of the fruit (see FIG. 11). FIG. 11 shows fluorescence images of exogenous zinc ions in (a) C2C12 and (b) NIH3T3 cells using CTMPA. Cells in this image were incubated in the absence (left) or presence (center) of ZnCl ₂ / pyrithione solution (2 μM) for 0.5 hours and then treated for 1 hour using 2 μM CTMPA. The cells in the right image were incubated with ZnCl ₂ / sodium pyrithione solution (2 μM) and then treated with 50 μM TPEN for 0.5 h. The treated cells were incubated with 2 μM CTMPA for 1 hour (λ _ex = 543 nm, scale bar = 20 μm). This observation indicates that CTMPA is cell-permeable and capable of detecting intracellular zinc ions.

Due to the fact that free zinc ions are released from intracellular metal proteins during apoptosis, CTMPA has also been used to detect this process. To induce apoptosis, C2C12 and NIH3T3 cells were incubated for 6 hours with 250 μΜ H ₂ O ₂ and subsequently treated with CTMPA for 1 hour. Analysis of the fluorescence microscopy images shows that cells incubated with H ₂ O ₂ show bright red fluorescence (see FIGS. 12 and 13). FIG. 12 shows fluorescence images of intact zinc ions in (a) C2C12 and (b) NIH3T3 cells released during apoptosis using CTMPA according to this Example, and FIG. 13 shows CTMPA according to this Example. Fluorescence images of intact zinc ions released in dead cells in (a) C2C12 and (b) NIH3T3 cells are shown. To confirm that the turn-on of fluorescence is the result of CTMPA's response to released zinc ions, H ₂ O ₂ -treated cells were treated with 50 μM TPEN for 0.5 h prior to treatment with the probe. Were incubated. In this case, the fluorescence intensity of the cells was significantly reduced (FIG. 12), indicating that the probe senses the zinc ions actually released during apoptosis. Figure 12 shows fluorescence imaging of intact zinc ions in (a) C2C12 and (b) NIH3T3 cells released during apoptosis using CTMPA. Cells were cultured in the absence (left) or presence (center) of 250 μM H ₂ O ₂ for 6 hours, causing apoptosis and then treated for 1 hour with 2 μM CTMPA. The cells in the right image were incubated for 6 hours using 250 μM H ₂ O ₂ , treated with 50 μM TPEN for 0.5 hour, and then incubated with 2 μM CTMPA for 1 hour (λ _ex = 543 nm, scale bar = 20 μm).

Intact Zn in zebrafish with CTMPA ²⁺ Detection of

A more interesting application of CTMPA is the detection of Zn ²⁺ in living organisms. Zebrafish are useful vertebrate models aimed at detecting fluorescence of various biologically related species and biomolecules. In conclusion, the applicability of CTMPA to tracking the distribution of intact zinc ions during zebrafish development has been studied. With this effort, zebrafish embryos at various stages of development (24, 36, 48, 72 and 96 hours post-fertilization (hpf) after exposure) were exposed to 2 μM CTMPA at 28 ° C. (see FIG. 14). . (A) 24 hours, (b) 36 hours, (c) 48 hours, (d) 72 hours, and (e) 96 hours after fertilization incubated with CTMPA for 1 hour according to this Example. Photo of fluorescence detection of intact zinc ions in zebrafish. When observed using different probes such as NBD-TPEA and ZTRS, a bright red fluorescence spot occurred at the bottom of the abdomen after 24 hpf of the embryo, and red dot bands consisting of multiple red spots were directed to the top of the abdomen after 48 hpf. Moved and then disappeared after 72 hpf (see FIG. 14). In addition, red spots developed around the egg yolk. To confirm that the fluorescence points were formed in the response of the probe to intact Zn ²⁺ in zebrafish, the embryos were exposed to about 100 μM TPEN for about 0.5 hours and then processed using CTMPA. Red fluorescence in zebrafish was hardly detectable in embryos that had been exposed to the TPEN pretreatment protocol (see FIG. 15), which means that the red dot is actually the result from endogenous zinc ions in the fish.

It has previously been shown that metallothioneins expressed in neurospheres distributed on the living surface play an important role in Zn ²⁺ homeostasis. Importantly, by using CTMPA, we were able to observe the fluorescence signal in zebrafish neurospheres without treatment with exogenous zinc ions (FIG. 14E). The fluorescence intensity of this probe in neurospheres of zebrafish was significantly reduced by pretreatment with TPEN, which supports the conclusion that the new probe can selectively detect intact zinc ions in neurospheres (FIG. 15). ). FIG. 16 is an image showing the distribution of neurospheres according to the grouped living bodies in (A) side and (B) dorsal regions according to the present embodiment, and the upper eye and the supraorbital region are in front of the visual cross (preoptic, PO). ), And supraorbital (SO) neurospheres. The caudal-cranial region includes the ear (otic, O), the occipital (OC), the dorsal (D), and the middle (MI) neurospheres, and the posterior (P) Neurospheres are placed in the trunk area. It should be noted that this phenomenon was not observed when using different zinc probes such as NBD-TPEA and ZTRS. Since CTMPA has a very high binding affinity for Zn ²⁺ , and CTMPA exhibits long wavelength luminescence that inhibits autofluorescence, these probes have a unique ability to detect intact zinc ions in zebrafish neurospheres. .

The above-mentioned studies lead to the development of CTMPA as a novel fluorescent Zn ²⁺ sensor with long wavelength emission maximum. With this effort, we demonstrated that CTMPA has high sensitivity and high selectivity for Zn ²⁺ relative to other metal ions, and a detection limit in the nanomolar range. The significant absorption and emission wavelength shifts that occur when CTMPA forms a complex with Zn ²⁺ make the sensor ideally suited for bioimaging of endogenous free zinc ions. This advantageous feature was demonstrated by using CTMPA for fluorescence detection of intact zinc ions in the zebrafish neurospheres. These interesting results mean that CTMPA will have a bright future in research aimed at understanding Zn ²⁺ biology.

The strategy devised by the inventors of the present invention is the high sensitivity and high selectivity, ratiometric cyanine-based metal ion sensor, which is dependent on the modulation of the conjugation length of the π-electron of the cyanine chromophore / fluorophore by metal ion coordination bonds. It may be applicable to the formation. In particular, an approach based on apparent changes in the π-electron system of cyanine dyes will serve as the basis for devising other types of cyanine-based fluorescent probes for in vivo imaging applications.

The above description of the present application is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present application. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present application is indicated by the following claims rather than the above description, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present application.

Claims (17)

1. Cyanine-based fluorescent probe comprising a compound represented by the following formula (1):

   [Formula 1]

   

   .
2. The method of claim 1,

   The cyanine-based fluorescent probe is used for the detection of zinc ions (Zn ²⁺ ), cyanine-based fluorescent probe.
3. The method of claim 1,

   The compound represented by Formula 1 is to react with zinc ions (Zn ²⁺ ) to form a complex, cyanine-based fluorescent probe.
4. The method of claim 3, wherein

   The compound represented by the formula (1) is a cyanine-based fluorescent probe that changes from blue to red at the maximum emission wavelength in response to the Zn ²⁺ .
5. The method of claim 4, wherein

   The compound represented by the formula (1) is a cyanine-based fluorescent probe that exhibits a short wavelength shift in the emission wavelength and the absorption wavelength by the reaction with the Zn ²⁺ .
6. The method of claim 4, wherein

   The compound represented by the formula (1) is a cyanine-based fluorescent probe that shows a short wavelength shift from 730 nm to 590 nm at the maximum emission wavelength by the reaction with Zn ²⁺ .
7. The method of claim 1,

   Pyridine and meso-substituted nitrogen in the compound represented by the formula (1) is a cyanine-based fluorescent probe that is coordinated by reacting with the Zn ²⁺ .
8. The method of claim 3, wherein

   Cyanine-based fluorescent probe that the compound represented by the formula (1) and the Zn ²⁺ forms a complex in a ratio of 1: 1.
9. The method of claim 1,

   The cyanine-based fluorescent probe detects endogenous zinc ions (Zn ²⁺ ) in vivo.
10. The method of claim 1,

    The cyanine-based fluorescent probe is to detect zinc ions within the nM range, cyanine-based fluorescent probe.
11. The detection method of zinc ion in vivo using the cyanine fluorescent probe of any one of Claims 1-10.
12. The method of claim 11,

    The detection method is to detect the distribution of endogenous zinc ions in vivo, detection method of zinc ions in vivo.
13. A method for preparing a cyanine-based fluorescent probe comprising reacting TMPA represented by Formula 2 with tricarbocyanide chloride:

    [Formula 2]

    

    .
14. The method of claim 13,

    The TMPA and the tricarbocyanide chloride is a method of producing a cyanine-based fluorescent probe is heated and reacted at 70 ℃ to 100 ℃ under argon atmosphere.
15. The method of claim 13,

    Method for producing a cyanine-based fluorescent probe further comprises purifying the cyanine-based fluorescent probe.
16. A fluorescent sensor comprising the cyanine-based fluorescent probe according to any one of claims 1 to 10.
17. Cyanine compound represented by the following formula (1):

    [Formula 1]

    

    .

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